Screening for novel human genes associated with CRE pathway activation with cell microarray.
Screening for novel human genes associated with CRE pathway activation with cell microarray.
复制标题
DOI：
10.1016/j.ygeno.2007.02.004
复制
发表时间：
2007-07
期刊：
影响因子：
4.4
通讯作者：
Linjie Tian;Pingzhang Wang;Jinhai Guo;Xinyu Wang;Weiwei Deng;Chenying Zhang;Dongxu Fu;Xia Gao;T. Shi;D. Ma
中科院分区：
文献类型：
--
作者：
Linjie Tian;Pingzhang Wang;Jinhai Guo;Xinyu Wang;Weiwei Deng;Chenying Zhang;Dongxu Fu;Xia Gao;T. Shi;D. Ma
在这项研究中，细胞微阵列技术被用来确定新的人类基因与CRE途径激活。通过反向转染，将含有全长cDNA的表达质粒与报告质粒pCRE-d2 EGFP共转染，以通过增强的绿色荧光蛋白（EGFP）表达来监测CRE途径的激活。在筛选的575个主要新基因中，22个与阴性对照相比表现出相对较高的EGFP荧光。在用包括顺式和反式荧光素酶测定的双荧光素酶报告系统进行功能验证后，22个基因中的4个（RNF 41、C8 orf 32、C6 orf 208和MEIS 3 P1）被确认为CRE途径激活剂。Western blot分析显示RNF 41可促进CREB磷酸化。这些结果证明了细胞微阵列技术与该报告系统的成功结合，以及该工具以高度平行的形式表征新基因功能的潜力。
In this study, cell microarray technology is used to identify novel human genes associated with CRE pathway activation. By reverse transfection, expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pCRE-d2EGFP to monitor the activation of the CRE pathway via enhanced green fluorescence protein (EGFP) expression. Of the 575 predominantly novel genes screened, 22 exhibited relatively higher EGFP fluorescence compared with a negative control. After a functional validation with a dual luciferase reporter system that included both cis- and trans-luciferase assays, 4 of the 22 genes (RNF41, C8orf32, C6orf208, and MEIS3P1) were confirmed as CRE-pathway activators. Western blot analysis revealed that RNF41 can promote CREB phosphorylation. These results demonstrate the successful combination of cell microarray technology with this reporting system and the potential of this tool to characterize functions of novel genes in a highly parallel format.